PB2202: FEATURES OF COLONY FORMING ABILITY OF BONE MARROW IN PATIENTS WITH NEUTROPENIA
Authors: M. Butolina, E. Poponina, O. Tselousova, E. Vaskina, A. Pestrikova
Journal: HemaSphere (2022)

Background: To study the proliferative and differentiating ability of hematopoietic precursors and to determine the hypoplastic variant of neutropenia, a cultural method is used. A special semi-liquid nutrient medium based on methylcellulose is used in this study. A certain amount of nucleated bone marrow cells is incubated for a fixed period. Colony forming units (CFU) make colonies in depth and on the surface of the medium. The number of colonies is proportional to the amount of the corresponding colony-forming cells in cell suspension. The colonies of each hematopoietic cell lines have characteristic morphological features which make it possible to distinguish and count them. Aims: Evaluating the colony-forming ability of the bone marrow using a culture method as part of the differential diagnosis of neutropenia of unknown origin. Methods: The colony-forming ability of the bone marrow was studied in 20 patients of children’s department of hematology clinic, surveyed in 2016-2021 (13 boys, 7 girls). Age ranged from 1.2 to 17 (median 13) years. The criterion for the neutropenia was the absolute number of neutrophils less than 1.5 thousand in 1 μl of peripheral blood. For cultivation, a nutrient medium based on methylcellulose (MethoCult Human No. 4434, StemCell Technologies, Canada) was used, inoculation was performed according to the standard method. Cultures are incubated at 37°C in 5% CO2.The proliferative potential and differentiation ability of bone marrow cells was determined by counting the number of corresponding colonies in a 14-day culture. The norm was considered for granulocyte-erythrocyte-macrophage-megakaryocytic colonies (CFU-GEMM) - 0-8 per 105 myelokaryocytes, granulocytic (CFU-G) - 7-35, macrophage (CFU-M) - 1-18, eosinophilic (CFU-Eo) – 0-20, respectively. The total number of colonies is normally 49-166. Results: The total number of colonies ranged from 6 to 158 (median 55). Of these, within the reference values - 55.0% of patients. The number of CFU-G was determined in the range from 1 to 45 (median - 4), in a number of patients the granulocytic cell line of the bone marrow was not weakened: in 25.0% of cases, a normal number of CFU-G was found, in 10.0% - increased. The number of CFU-M ranged from 3 to 67 (median 21.5); an increased number of macrophage precursors was observed in 60.0% of neutropenic patients. A direct correlation between the number of CFU-G and CFU-M was determined, Spearman’s coefficient (r) 0.64 (p=0,003). The increased activity of the granulocytic cell line of hematopoiesis was accompanied by a high number of CFU-M. In all patients, the number of CFU-GEMM (0-1, median - 0) and CFU-Eo (0-10, median - 1.5) was determined within the normal range. Summary/Conclusion: Neutropenia is a group of conditions with different etiology and pathogenesis. The culture method can be used for the differential diagnosis of neutropenia of unknown origin. Correlation of the number of granulocytic and macrophage precursors in the bone marrow in patients with neutropenia can be due to active hematopoiesis maybe because of cytokine stimulation.